Abstract Cryptococcosis is a deadly fungal disease that accounts for over 15% HIV/AIDS related deaths. Treatment options for cryptococcosis are limited. Currently available antifungal drugs are either highly toxic (polyenes) or exert a fungistatic effect (triazoles), necessitating long treatment regimens and leaving open the avenue for emergence of drug resistance. The third major antifungal drug class, the echinocandins, show low toxicity and are fungicidal against several other prevalent fungal pathogens. However, Cryptococci are resistant to echinocandins and the mechanisms of this resistance remain unknown. We recently reported that loss of lipid flippase, the enzyme responsible for maintaining the asymmetry of membrane lipid bilayers and normal intracellular vesicle trafficking, sensitizes C. neoformans to caspofungin, a drug of the echinocandin class, as well as to several triazoles. We also found that lipid flippase was essential for virulence in a murine model of cryptococcosis and sensitized C. neoformans to killing by macrophages, suggesting that it may be a novel antifungal drug target. In this project, we propose to decipher the mechanism of lipid flippase in cryptococcal echinocandin resistance and to conduct proof-of-principle studies inhibiting flippase function in C. neoformans. In the first Aim, we will test two related, non-mutually exclusive hypotheses regarding the role of lipid flippase in drug resistance: (1) that loss of lipid flippase changes membrane structure, e.g. PS distribution on membrane, to promote the interaction of caspofungin with its target ?-1,3-D-glucan synthase (Fks1), and (2) that in the absence of lipid flippase certain drug resistance pathways, such as calcineurin pathway are compromised, disrupting cellular calcium homeostasis and inducing killing by drugs. We will test these hypotheses by employing a host of cellular, molecular, biochemical, and genetic approaches. In the second Aim, we propose to develop an antibody Fab fragment against the extracellular loop of lipid flippase, which is essential for flippase function, and to test its ability to sensitize C. neoformans to antifungal drugs and to killing by macrophages. The region of lipid flippase targeted by this antibody-based approach has low sequence homology to its human counterpart, and our preliminary studies showed that an antibody raised against this region is fungal-specific. The success of this study will lead to a better understanding of lipid flippase mediated drug resistance in C. neoformans, which could help expand the use of echinocandin drugs against Cryptococci and other resistant fungal pathogens. Furthermore, generation of flippase inhibitory antibodies will provide a valuable research tool and may lead to future development of novel combination therapy approaches. Finally, successful development of antibody- based inhibitors could open a new avenue of research and drug development against other membrane proteins in fungi and bacteria.